CN116059325A - immune inducer - Google Patents

Abstract

The present invention aims to find a novel peptide useful as an active ingredient of a therapeutic or prophylactic agent for cancer, and to provide a use of the polypeptide as an immunity inducer. An immunity inducer comprising (a) a polypeptide comprising the amino acids represented by SEQ ID Nos. 3 to 45 and (b) a polypeptide obtained by deleting, substituting, inserting or adding 1 to several amino acids to the polypeptide of (a) as an active ingredient, which is useful as a therapeutic or prophylactic agent for cancer.

Description

immune inducer

This application is a divisional application of a patent application with an application date of March 1, 2017, an application number of 201780013955.2, and an invention title of "Immunity Inducing Agent".

technical field

The present invention relates to a novel immunity-inducing agent useful as an active ingredient of a drug for treating or preventing cancer.

Background technique

SCD1 (stearyl-CoA desaturase 1, stearoyl-CoA desaturase 1) protein is a protein that introduces a double bond at the C9-C10 position of saturated fatty acid.

The SCD1 protein has been suggested to be associated with cancer pathogenesis. For example, Non-Patent Documents 1 and 2 disclose that expression increases in various cancers such as liver cancer, esophageal cancer, and colorectal cancer. When the function of SCD1 is blocked by siRNA or a low-molecular-weight inhibitory compound, the proliferation of cancer cells is inhibited. Apoptosis is induced and established tumors shrink.

On the other hand, Patent Document 1 discloses that the SCD1 protein has immunity-inducing activity against cancer cells, and therefore is useful for the treatment and prevention of cancer. However, Patent Document 1 does not disclose information on peptides capable of binding to MHC molecules.

prior art literature

patent documents

Patent Document 1: WO2012/157736

non-patent literature

Non-Patent Document 1: Igal RA.Carcinogenesis.Sep; 31(9):1509-15(2010)

Non-Patent Document 2: Chen L.Sci.Rep.6,19665(2016)

Contents of the invention

The problem to be solved by the invention

The object of the present invention is to find a novel polypeptide useful as an active ingredient of a drug for treating or preventing cancer, and to provide a use of the polypeptide as an immunity-inducing agent.

Another object of the present invention is to provide isolated antigen-presenting cells comprising a complex of the polypeptide and MHC molecules, isolated T cells selectively binding to the complex of the polypeptide and MHC molecules, and their cancers. Curative or preventive medicine.

method used to solve the problem

The inventors of the present application conducted in-depth research, and as a result obtained the following insight: the human SCD1 protein composed of the amino acid sequence shown in SEQ ID NO: 2 is effective in malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, It is specifically expressed in tissues or cells of gastric cancer, malignant brain tumor, esophageal cancer and lung cancer. In addition, it was found that a partial peptide present in a specific region of the SCD1 protein has the ability to be presented by antigen-presenting cells to activate and proliferate T cells specific to the polypeptide (immunity-inducing activity), and that the immune-inducing activity has an effect on cancer. Useful for treatment or prevention. Based on these results, it has been found that this polypeptide can be used as an active ingredient of an immunity-inducing agent for the treatment and/or prevention of cancer, and that the antigen-presenting cells contacted with the peptide and the T cells contacted with the antigen-presenting cells are also effective against cancer. It is useful for treatment or prevention, and thus the present invention has been accomplished.

That is, the present invention has the following features (1) to (12).

(1) An immune-inducing agent, as an active ingredient, containing: at least one polypeptide selected from the polypeptide group described in (a) or (b) below and having immune-inducing activity, or containing at least one polypeptide encoding any one of the polypeptides A polynucleotide and a recombinant vector capable of expressing the polypeptide in vivo,

(a) 7 consecutive amino acids in the region of 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence shown in SEQ ID NO: 2 Polypeptides composed of the above amino acids,

(b) A polypeptide having one to several amino acids deleted, substituted, inserted or added to the amino acid sequence of any one of the polypeptides described in (a).

(2) The immune-inducing agent according to (1), wherein the polypeptide having immune-inducing activity is capable of binding to MHC class I molecules.

(3) The immune-inducing agent according to (2), wherein the polypeptide having immune-inducing activity is any polypeptide selected from the polypeptide group described in (c) to (e) below,

(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3-36,

(d) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (c),

(e) A polypeptide comprising the polypeptide described in (c) or (d) as a partial sequence.

(4) The immunity-inducing agent according to (1), wherein the polypeptide having immunity-inducing activity is capable of binding to MHC class II molecules.

(5) The immune-inducing agent according to (4), wherein the polypeptide having immune-inducing activity is any polypeptide selected from the group of polypeptides described in (f) to (h) below,

(f) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 37-45,

(g) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (f),

(h) A polypeptide comprising the polypeptide described in (f) or (g) above as a partial sequence.

(6) The immunity-inducing agent according to any one of (1) to (5), which is used as an active ingredient of a drug for treating or preventing cancer.

(7) The immunity-inducing agent according to (6), wherein the cancer expresses the SCD1 protein.

(8) The immunity-inducing agent according to (6) or (7), wherein the cancer is malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, gastric cancer, malignant brain tumor, esophageal cancer or lung cancer.

(9) The immunity-inducing agent according to any one of (1) to (8), further comprising an immunity-enhancing agent.

(10) An isolated antigen-presenting cell comprising a complex of the polypeptide having immune-inducing activity described in (1), (3) or (5) and MHC molecules.

(11) An isolated T cell capable of selectively binding to the complex of the polypeptide having immune-inducing activity described in (1), (3) or (5) and MHC molecule.

(12) Any polypeptide selected from the polypeptide group described in (a) or (b) below and having immune-inducing activity,

(a) 7 consecutive amino acids in the region of 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence shown in SEQ ID NO: 2 A polypeptide with immune-inducing activity composed of the above amino acids,

(b) A polypeptide having one to several amino acids deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (a).

(13) A drug for the treatment or prevention of cancer, containing as an active ingredient one or more species selected from the group consisting of the following (i) to (iv),

(i) At least one polypeptide selected from the polypeptide group described in (a) or (b) below and having immune-inducing activity:

(a) 7 consecutive amino acids in the region of 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence shown in SEQ ID NO: 2 Polypeptides composed of the above amino acids,

(b) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of any polypeptide described in (a);

(ii) a recombinant vector comprising at least one polynucleotide encoding any of the polypeptides and capable of expressing the polypeptide in vivo;

(iii) an isolated antigen-presenting cell comprising a complex of any of said polypeptides with an MHC molecule; and

(iv) isolated T cells specific for any of said polypeptides.

(14) The drug for treating or preventing cancer according to (13), wherein the polypeptide having immune-inducing activity is at least one polypeptide selected from the polypeptide group described in (c) to (h) below:

(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3-36,

(d) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (c),

(e) a polypeptide comprising the polypeptide described in (c) or (d) as a partial sequence,

(f) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 37-45,

(g) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (f),

(h) A polypeptide comprising the polypeptide described in (f) or (g) above as a partial sequence.

(15) The drug for treating or preventing cancer according to (13) or (14), wherein the cancer expresses the SCD1 protein.

(16) A method for treating or preventing cancer, comprising administering one or more steps selected from the group consisting of the following (i) to (iv) to a subject animal in need of cancer treatment or prevention,

(i) At least one polypeptide selected from the polypeptide group described in (a) or (b) below and having immune-inducing activity:

(a) 7 consecutive amino acids in the region of 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence shown in SEQ ID NO: 2 Polypeptides composed of the above amino acids,

(b) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of any polypeptide described in (a);

(ii) a recombinant vector comprising at least one polynucleotide encoding any of the polypeptides and capable of expressing the polypeptide in vivo;

(iii) an isolated antigen-presenting cell comprising a complex of any of said polypeptides with an MHC molecule; and

(iv) isolated T cells specific for any of said polypeptides.

(17) The method according to (16), wherein the polypeptide having immune-inducing activity is at least one polypeptide selected from the polypeptide group described in (c) to (h) below:

(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3-36,

(d) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (c),

(e) a polypeptide comprising the polypeptide described in (c) or (d) as a partial sequence,

(f) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 37-45,

(g) a polypeptide in which 1 to several amino acids are deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (f),

(h) A polypeptide comprising the polypeptide described in (f) or (g) above as a partial sequence.

(18) The method according to (16) or (17), wherein the cancer expresses the SCD1 protein.

This specification includes the disclosure of Japanese Patent Application No. 2016-040364 which is the basis of priority of this application.

The effect of the invention

According to the present invention, a novel immunity-inducing agent useful as an active ingredient of a drug for treating or preventing cancer is provided.

In addition, as will be concretely demonstrated in the examples described below, the polypeptides used in the present invention can induce immune cells that kill cancer cells, and can shrink or shrink cancers that have already developed. Furthermore, the peptides used in the present invention can enhance the induction of immune cells that kill cancer cells, and shrink or shrink cancers that have already developed. Therefore, the polypeptide of the present invention is useful as an active ingredient of a drug for treating or preventing cancer.

Description of drawings

Fig. 1 is a graph showing the expression pattern of SCD1 gene in human tumor tissues or cancer cell lines. Reference number 1: shows the expression profile of the human SCD1 gene. Reference No. 2: An expression map of the GAPDH gene, which is a human housekeeping gene, is shown.

Fig. 2 is a diagram showing that CD8-positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 3 to 23 recognize the complex of the polypeptide and HLA-A0201 and produce IFN-γ. In the figure, bands 4 to 24 on the horizontal axis show the IFN-γ production ability of HLA-A0201-positive CD8-positive T cells stimulated by dendritic cells pulsed with the polypeptides represented by the amino acid sequences of SEQ ID NOs. 3-23, respectively. Band 1 shows the result of the above treatment without adding the polypeptide (Mock), band 2 shows the result of adding the negative control polypeptide shown in sequence number 46 outside the scope of the present invention to carry out the above treatment, and band 3 shows the addition of sequence number 2 The amino acid sequence shown consists of the full-length SCD1 protein as a result of the above treatments.

Fig. 3 is a diagram showing that CD8-positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 24 to 36 recognize the complex of the polypeptide and HLA-A24 and produce IFN-γ. In the figure, bands 4 to 16 on the horizontal axis show the IFN-γ production ability of HLA-A24-positive CD8-positive T cells stimulated by dendritic cells pulsed with the polypeptide represented by the amino acid sequence of 24-36, respectively. Lane 1 shows the result of the above treatment without adding the polypeptide (Mock), Lane 2 shows the result of the above treatment with the addition of a negative control peptide shown in Serial No. 47 outside the scope of the present invention, and Lane 3 shows the addition of The results of the above treatment of the full-length SCD1 protein composed of the amino acid sequence shown in 2.

Fig. 4A is a graph showing the cytotoxic activity of CD8-positive T cells specific to each polypeptide consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 23 against cancer cells. In the figure, bands 4 to 24 on the horizontal axis show the cytotoxic activity of HLA-A0201 positive CD8 positive T cells against U251 cells induced by the polypeptides represented by the amino acid sequences of SEQ ID NOs. 3 to 23, respectively. Band 1 shows the cytotoxic activity of CD8-positive T cells (Mock) induced without adding polypeptide, Band 2 shows the cytotoxic activity of CD8-positive T cells induced by negative control polypeptide (SEQ ID NO: 46), and Band 3 shows the cytotoxic activity of CD8-positive T cells induced by The cytotoxic activity of CD8-positive T cells induced by the full-length SCD1 protein composed of the amino acid sequence shown in SEQ ID NO: 2.

Fig. 4B is a graph showing the cytotoxic activity of CD8-positive T cells specific to each polypeptide consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 23 against cancer cells. In the figure, bands 4 to 24 on the horizontal axis show the cytotoxic activity of HLA-A0201-positive CD8-positive T cells against SK-Hep-1 cells induced by the polypeptides represented by the amino acid sequences of SEQ ID NOs. 3-23, respectively. Band 1 shows the cytotoxic activity of CD8 positive T cells (Mock) induced without adding polypeptide, band 2 shows the cytotoxic activity of CD8 positive T cells induced by negative control polypeptide (SEQ ID NO. 46), and band 3 shows the cytotoxic activity of CD8 positive T cells induced by the sequence The cytotoxic activity of CD8-positive T cells induced by the full-length SCD1 protein composed of the amino acid sequence shown in No. 2.

Fig. 5A is a graph showing the cytotoxic activity of CD8-positive T cells specific to each polypeptide consisting of the amino acid sequences represented by SEQ ID NOs: 24 to 36 against cancer cells. Bands 4 to 16 on the horizontal axis show the cytotoxic activity of HLA-A24-positive CD8-positive T cells stimulated with the polypeptides represented by the amino acid sequences of SEQ ID NOs: 24-36 against SW480 cells, respectively. Lane 1 shows the cytotoxic activity of CD8-positive T cells (Mock) induced without adding the polypeptide, Reference No. 2 shows the cytotoxic activity of CD8-positive T cells induced by using a negative control polypeptide (SEQ ID NO: 47), and Band 3 shows the cytotoxic activity of CD8-positive T cells induced by using Cytotoxic activity of CD8-positive T cells induced by the SCD1 protein consisting of the amino acid sequence shown in SEQ ID NO: 2.

Fig. 5B is a graph showing the cytotoxic activity of CD8-positive T cells specific to each peptide consisting of the amino acid sequences shown in SEQ ID NOs: 24 to 36 against cancer cells. Bands 4 to 16 on the horizontal axis show the cytotoxic activity against ZR-75-1 cells of HLA-A24-positive CD8-positive T cells stimulated with the polypeptides represented by the amino acid sequences of SEQ ID NOs: 24-36, respectively. Lane 1 shows the cytotoxic activity of CD8-positive T cells (Mock) induced without adding the polypeptide, Reference No. 2 shows the cytotoxic activity of CD8-positive T cells induced by using the negative control polypeptide (SEQ ID NO: 47), and Band 3 shows the cytotoxic activity of CD8-positive T cells induced by using Cytotoxic activity of CD8-positive T cells induced by the SCD1 protein consisting of the amino acid sequence shown in SEQ ID NO: 2.

Fig. 6 is a diagram showing that CD4-positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 37 to 45 recognize the complex of the polypeptide and HLA-DRB1*04 to produce IFN-γ. Lanes 4 to 12 show the IFN-γ production ability of HLA-DRB1*04-positive CD4-positive T cells stimulated by dendritic cells pulsed with the polypeptides represented by the amino acid sequences of SEQ ID NOs: 37-45, respectively. Band 1 shows the result of Mock when the above-mentioned treatment is performed without adding the polypeptide, band 2 shows the result of the above-mentioned treatment with the addition of the negative control polypeptide shown in SEQ ID No. The amino acid sequence shown consists of the full-length SCD1 protein as a result of the above treatments.

Detailed ways

＜Polypeptide＞

In the present invention, "polypeptide" refers to a molecule in which a plurality of amino acids are formed by peptide bonds. Not only polypeptide molecules having a large number of amino acids but also low-molecular-weight molecules (oligopeptides) having a small number of amino acids are included in the polypeptide of the present invention.

The polypeptide constituting the immunity-inducing agent of the present invention includes at least one polypeptide selected from the polypeptide group described in (a) or (b) below and having an immunity-inducing activity.

(a) In the human SCD1 protein composed of the amino acid sequence shown in SEQ ID NO: 2, when the starting methionine is the first position, the 34-50 (17 amino acids), 69-148 (80 amino acids) amino acids), 178-195 (18 amino acids), 207-242 (36 amino acids), 247-280 (34 amino acids), 296-332 (37 amino acids) amino acid peptides

(b) A polypeptide having one to several amino acids deleted, substituted, inserted or added to the amino acid sequence of the polypeptide described in (a) above.

In addition, "consisting of an amino acid sequence" in the present invention means that the amino acid residues are arranged in this order. Therefore, for example, "a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2" refers to having the amino acid sequence of Met Asp ProAla···(omitted in the middle)···Tyr Lys Ser Gly shown in SEQ ID NO: A polypeptide of 359 amino acid residues. In addition, in this specification, for example, "the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2" may be abbreviated as "the polypeptide of SEQ ID NO: 2". The same applies to the expression "consisting of a base sequence".

In addition, "immunity-inducing activity" in the present invention refers to the ability to activate and proliferate T cells that react with cancer cells expressing the SCD1 protein. Specifically, it means: the IFN-γ production ability of cytotoxic T cells and/or helper T cells stimulated by SCD1 protein or its partial polypeptides is higher than that of unstimulated control T cells; The cytotoxic activity of stimulated cytotoxic T cells to cancer cells expressing SCD1 protein is higher than that of unstimulated control T cells; helper T cells stimulated by SCD1 protein or its partial polypeptides are more active than unstimulated controls The T cells can enhance the cytotoxic activity of the cytotoxic T cells; or the cytotoxic T cells or helper T cells stimulated by the SCD1 protein or its partial polypeptide proliferate better than the unstimulated control T cells.

Proliferation of cells can be confirmed by visual observation, cell count under a microscope, flow cytometry, incorporation of tritiated thymidine in the medium into cells, and the like. In addition, the measurement of IFN-γ production ability can be confirmed using, for example, a known enzyme-linked immunospot (ELISpot) assay or the like. Specifically, for example, as described in the Examples described below, first, T cells are combined with a polypeptide (in the present invention, the SCD1 protein or a partial polypeptide thereof) that should be evaluated for immune-inducing activity, and peripheral blood mononuclear cells (hereinafter expressed as " PBMC") antigen-presenting cells are co-cultured so that T cells are in contact with antigen-presenting cells presenting the polypeptide to be evaluated. Next, IFN-γ produced by T cells was measured using an antibody specific for IFN-γ. Thereby, the number of immune cells in the T cells can be measured. The immune-inducing activity can be evaluated from the measurement results.

In addition, for the measurement of cytotoxic activity, for example, after co-cultivating T cells with a polypeptide to be evaluated for cytotoxic activity (microSCD1 protein or a partial polypeptide thereof in the present invention) and antigen-presenting cells derived from PBMC, it is investigated in vitro whether or not the inhibitory activity is exhibited. The ability to proliferate tumor cells or the ability to kill tumor cells (hereinafter referred to as "cytotoxic activity") was evaluated. The contact between T cells and antigen-presenting cells can be achieved by co-cultivating both in a liquid medium as will be described later. The cytotoxic activity can be measured by a known method called ⁵¹ Cr release assay described in Int. J. Cancer, 58: P317, 1994, for example.

Administration of the above-mentioned induced T cells to a cancer-bearing organism can shrink or regress tumors through the cytotoxic activity of the T cells. Therefore, the above-mentioned immunity-inducing activity can also be evaluated as the ability to inhibit the proliferation of cancer cells, or shrink or eliminate cancerous tissues (tumors) (hereinafter referred to as "anti-tumor activity").

When the above-mentioned polypeptide is used for the treatment or prevention of cancer, although it is not particularly limited, the evaluation of the immune-inducing activity is preferably based on cytotoxic activity or antitumor activity as an index.

As is well known in the art, any polypeptide having about 7 or more amino acid residues can contain an epitope, can exhibit antigenicity and immunogenicity, and can have immunity-inducing activity, so it can be used as the immunity-inducing agent of the present invention.

Therefore, the polypeptide of the above (a) is composed of 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence shown in SEQ ID NO: 2. A polypeptide consisting of more than 7 consecutive amino acids, preferably more than 8, 9 or 10 consecutive amino acids in the region, and having immune-inducing activity. Particularly preferred are polypeptides having the amino acid sequences shown at positions 34-50, 69-148, 178-195, 207-242, 247-280, and 296-332 in the amino acid sequence shown in SEQ ID NO: 2. .

As the principle of immune induction by administering cancer antigen polypeptides, it is known that the polypeptides are taken up into antigen-presenting cells, then decomposed by peptidases in the cells to form small fragments, and then the fragmented antigen peptides are presented to the antigen-presenting cells. on the surface of the cells. Cytotoxic T cells and the like recognize antigens presented on the cell surface and selectively kill cancer cells that present the antigens on the cell surface. In addition, it is known that helper T cells recognize antigens presented on the surface of antigen-presenting cells and promote the induction of cytotoxic T cells that selectively kill cancer cells that present the antigens on the cell surface. The size of the antigen polypeptide presented on the surface of the antigen-presenting cell is relatively small, and the number of amino acids is about 7 to 30. Therefore, from the viewpoint of being presented on antigen-presenting cells, as the polypeptide of (a) above, the 34th to 50th, 69th to 148th, 178th to 195th, 207th to About 7 to 30 consecutive amino acid sequences shown at positions 242, 247 to 280, and 296 to 332. It is sufficient if the polypeptide consists of about 8 to 30, about 9 to 30, or about 9 to 25 amino acids. These relatively small polypeptides are sometimes not taken up into antigen-presenting cells, but are directly presented on the cell surface on antigen-presenting cells.

In addition, polypeptides taken up by antigen-presenting cells are cleaved at random positions by peptidases in the cells to generate various polypeptide fragments, and these polypeptide fragments are presented on the surface of antigen-presenting cells. 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence of the amino acid sequence, the peptides can be decomposed in antigen-presenting cells , it is bound to produce a polypeptide fragment effective in inducing immunity through antigen-presenting cells. Therefore, large-sized polypeptides can also be used for the induction of immunity through antigen-presenting cells. For example, the number of amino acids in the polypeptide can be 30 or more, preferably 40 or more, more preferably 50 or more, and still more preferably 100 or more.

Furthermore, the polypeptide of the present invention can be obtained from 8 to 25, preferably 9 to 24, more preferably Checking media for epitope peptides consisting of 9 to 23 amino acids, such as HLA Peptide Binding Predictions (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.htmL) of Bioinformatics&Molecular Analysis Selection (BIMAS), SYFPEⅠTHⅠto check , to screen for peptides that can become epitope peptides, thereby obtaining. Specifically, the polypeptide of the present invention consists of the 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 regions in the amino acid sequence shown in SEQ ID NO: 2 A polypeptide consisting of more than 7 consecutive amino acids. For example, the polypeptides of the present invention include polypeptides represented by SEQ ID NOs. 3 to 45, or polypeptides consisting of the amino acid sequences represented by SEQ ID NOs. 3 to 45 as partial sequences and having 10 to 30 amino acid residues. peptide. Among the polypeptides represented by SEQ ID NOs 3-45, and the polypeptides comprising the amino acid sequences represented by SEQ ID NOs 3-45 as partial sequences and having 10-30 amino acid residues, SEQ ID NOs 3-36 The immune-inducing activity of the polypeptide shown is exerted by binding to MHC class I molecules, and the immune-inducing activity of the polypeptides shown in SEQ ID Nos. 37-45 is exerted by binding to MHC class II molecules.

On the other hand, the polypeptide of (b) above is a polypeptide in which one or several amino acid residues are substituted, deleted, inserted and/or added to the polypeptide of (a) above, and has immune-inducing activity. For example, polypeptides of the present invention include polypeptides in which one to several amino acids are deleted, substituted, inserted, or added to polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 45.

The "number" in "several" in this specification represents an integer of 2-10, Preferably it is an integer of 2-6, More preferably, it is an integer of 2-4, More preferably, it is an integer of 2 or 3.

It is generally believed that the change of one or several amino acids in a certain polypeptide does not affect the function of the original polypeptide, and sometimes even strengthens the desired function of the original polypeptide. In fact, it is known that an altered peptide consisting of an amino acid sequence in which one or several amino acid residues are changed (i.e., substituted, deleted, added and/or inserted) compared with the original amino acid sequence maintains the biological activity of the original peptide ( Mark et al., 1984, Proc Natl Acad Sci USA, 81:5662-5666, Zoller and Smith, 1982, Nucleic Acids Res.10: 6487-6500, Dalbadie-McFarland et al., 1982, Proc Natl Acad Sci USA.79 :6409-6413). Therefore, since the polypeptide of (b) above can also exhibit immunity-inducing activity, it can be used in the preparation of the immunity-inducing agent of the present invention.

In addition, the 20 amino acids constituting natural proteins can be classified as neutral amino acids with low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp) are grouped into groups with similar properties , it is known that the properties of the replacement polypeptides in each group do not change much. Therefore, when substituting the amino acid residues in the polypeptide (a) of the present invention, it is highly likely that the immunity-inducing activity can be maintained by substituting within each of these groups, which is therefore preferable.

In addition, the polypeptide of the above (b) may be the same as 34-50, 69-148, 178-195, 207-242, 247-280, 296-332 in the amino acid sequence shown in SEQ ID NO: 2. Any of the polypeptides consisting of 7 or more consecutive amino acids in the region of the sequence number 3 to 45, for example, has 90% or more, preferably 95% or more, more preferably 98% or more, and further A polypeptide having an amino acid sequence identity of 99% or more or 99.5% or more and having immune-inducing activity is preferred.

The "identity" of the amino acid sequence (or base sequence) in this specification means that the amino acid residues (or bases) of the two amino acid sequences (or base sequences) to be compared are as consistent as possible. Amino acid sequence (or base sequence) alignment, the value obtained by dividing the number of consistent amino acid residues (or the number of bases) by the total number of amino acid residues (or the number of bases) expressed in percentage. When performing the above arrangement, gaps are appropriately inserted in one or both of the two sequences to be compared as necessary. Such alignment can be performed using known programs such as BLAST, FASTA, and CLUSTALW. When a gap is inserted, the above-mentioned total number of amino acid residues becomes the number of residues counted by counting one gap as one amino acid residue. Thus, when the total number of amino acid residues counted is different between the two sequences to be compared, the sequence identity (%) is obtained by dividing the number of identical amino acid residues by the total number of amino acid residues of the longer sequence. Calculated.

When used in connection with cancer treatment or prevention, the polypeptide of the present invention should preferably be presented on the surface of cells or exosomes as a complex with various types of HLA. Therefore, the polypeptide of the present invention is preferably selected from a peptide that not only has immune-inducing activity but also has high binding affinity to various types of HLA. Therefore, the peptide may be modified by amino acid residue substitution, insertion, deletion, and/or addition to improve binding affinity. In addition to peptides that are naturally presented, the regularity of the sequences of peptides presented by binding to various types of HLA is known (J Immunol, 1994, 152: 3913; Immunogenetics, 1995, 41: 178; J Immunol, 1994, 155:4307), changes based on such regularity can be introduced into the immunogenic peptides of the present invention. For example, in order to improve HLA-A24 binding affinity, it may be desirable to replace the amino acid at the second position from the N-terminal with leucine or methionine, and/or replace the amino acid at the C-terminal with valine or leucine replace. Therefore, a peptide obtained by replacing the amino acid at the second position from the N-terminal of the peptide having the amino acid sequence of SEQ ID NO: 24 to 36 with leucine or methionine, and/or the C-terminal amino acid with valine or leucine. Peptides obtained by amino acid substitutions are included within the scope of the present invention.

Substitution can be introduced not only at the position of the terminal amino acid but also at a position capable of TCR recognition of the peptide. Several studies have confirmed that amino acid substitutions of peptides have equal or better immune-inducing activity than the original peptide, such as CAP1, p53(264-272), Her-2/neu(369-377), or gp100(209 -217) (Zaremba et al.1997, CancerRes.57:4570-4577, T.K.Hoffmann et al.2002, JImmunol.168(3):1338-47, S.O.Dionne et al.2003, CancerImmunol immunoother.52:199-206 , and S.O.Dionne et al. 2004, Cancer Immunology, Immunotherapy, 53:307-314).

In addition to the above-mentioned changes, the polypeptide of the present invention can also be linked to other substances as long as the resulting linked polypeptide maintains the necessary immunity-inducing activity of the original peptide. Examples of other substances are not limited, but include peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. Peptides may contain changes such as glycosylation, side chain oxidation, or phosphorylation, provided that the biological activity of the original peptide is not impaired by the changes. These kinds of changes can be made to confer additional functions (eg, targeting and delivery functions) or to stabilize the polypeptide. For example, techniques for introducing D-amino acids, amino acid mimetics or unnatural amino acids to improve the in vivo stability of polypeptides are well known in the art, and this concept can also be applied to the polypeptides of the present invention. The stability of a polypeptide can be determined by several methods. For example, peptidases, and various biological media such as human plasma and serum can be used to test stability (see, for example, Verhoef et al., 1986, Eur J Drug Metab Pharmacokin, 11: 291-302).

Furthermore, the polypeptide of the present invention can be linked to other peptides via a spacer or a linker. Examples of other peptides are not limited, and epitope peptides derived from other polypeptides are included. Alternatively, two or more polypeptides of the present invention may be linked via a spacer or a linker. The peptides linked via a spacer or a linker may be the same or different from each other. The types of the spacer and linker are not particularly limited, and include those made of peptides, more preferably those made of peptides having one or more cleavage sites that can be cleaved by enzymes such as peptidases, proteases, and proteasomes. Examples of linkers or spacers are not limited, and AAY (P.M.Daftarian et al., J Trans Med, 2007, 5:26), AAA, NKRK (R.P.M.Sutmuller et al., J Immunol.2000, 165:7308- 7315), or one to several lysine residues (S.Ota et al., 2002, CanRes.62:1471-1476, K.S.Kawamura et al., 2002, JImmunol.168:5709-5715). The present invention contemplates polypeptides linked to other peptides via spacers or linkers.

Where the polypeptides of the present invention contain cysteine residues, these polypeptides have a tendency to form dimers via disulfide bonds between the SH groups of the cysteine residues. Therefore, dimers of polypeptides are also included in the polypeptides of the present invention.

The polypeptides of the present invention can be prepared using known techniques. It can be synthesized by a chemical synthesis method such as the Fmoc method (fluorenylmethoxycarbonyl method) and the tBoc method (tert-butoxycarbonyl method). In addition, it can also be synthesized by a conventional method using various commercially available peptide synthesizers.

Furthermore, a polynucleotide encoding the above-mentioned polypeptide can also be prepared by using known genetic engineering techniques, inserted into an expression vector and introduced into a host cell, and the target polypeptide is produced in the host cell to obtain the target polypeptide. When the polypeptide of interest is obtained from host cells, it may be purified or isolated so that it does not substantially contain other native host cell proteins and their fragments, or any other chemical substances.

Polynucleotides encoding the above-mentioned polypeptides can be easily prepared by conventional methods using known genetic engineering techniques or commercially available nucleic acid synthesizers. For example, DNA having the nucleotide sequence of SEQ ID NO: 1 can be prepared by performing PCR with a pair of primers designed to amplify the nucleotide sequence described in SEQ ID NO: 1 using human chromosomal DNA or a cDNA library as a template. The PCR reaction conditions can be appropriately set, for example, a reaction process consisting of 94°C for 30 seconds (denaturation), 55°C for 30 seconds to 1 minute (annealing), and 72°C for 2 minutes (extension) as one cycle, For example, conditions such as 72° C. reaction for 1 minute after 30 cycles are performed, but are not limited thereto. Also, by preparing appropriate probes or primers based on the nucleotide sequence and amino acid sequence information shown in SEQ ID NO: 1, and using them to screen a human or the like cDNA library, desired DNA can be isolated. The cDNA library is preferably prepared from cells, organs or tissues expressing the protein of SEQ ID NO:2. Operations such as the preparation of the above-mentioned probes or primers, the construction of cDNA libraries, the screening of cDNA libraries, and the cloning of target genes are known to those skilled in the art, for example, according to Green, M.R. and Sambrook, J., 2012, Molecular Cloning : A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, or Current Protocol Molecular Biology: www.currentprotocols.com The method described in etc. implementation. DNA encoding the polypeptide of (a) above can be obtained from the DNA thus obtained. In addition, since the codons encoding each amino acid are known, the base sequence of a polynucleotide encoding a specific amino acid sequence can be easily determined. Therefore, since the nucleotide sequence of the polynucleotide encoding the polypeptide of (b) can also be easily determined, such a polynucleotide may be synthesized by a conventional method using a commercially available nucleic acid synthesizer.

As the above-mentioned host cell, any cell may be used as long as it is a cell capable of expressing the above-mentioned polypeptide. Examples of prokaryotic cells include Escherichia coli, etc., and examples of eukaryotic cells include monkey kidney cells COS1, Chinese hamster ovary cells, CHO, etc. Mammalian cultured cells, budding yeast, fission yeast, silkworm cells, Xenopus egg cells, etc., but not limited to these.

When prokaryotic cells are used as host cells, expression vectors having an origin capable of replication in prokaryotic cells, a promoter, a ribosome binding site, a DNA cloning site, a terminator, and the like are used. Examples of expression vectors for Escherichia coli include pUC system, pBluescriptII, pET expression system, pGEX expression system, and the like. The DNA encoding the above-mentioned polypeptide can be inserted into such an expression vector, and the prokaryotic host cell can be transformed with the vector, and the obtained transformant can be cultured to express the polypeptide encoded by the above-mentioned DNA in the prokaryotic host cell. In this case, the polypeptide may also be expressed as a fusion protein with another protein.

When eukaryotic cells are used as host cells, an expression vector for eukaryotic cells having a promoter, a splicing region, a poly A addition site, and the like is used as an expression vector. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 and the like. In the same manner as above, DNA encoding the above-mentioned polypeptide can be inserted into such an expression vector, and eukaryotic host cells can be transformed with the vector, and the obtained transformants can be cultured to express the polypeptide encoded by the above-mentioned DNA in the eukaryotic host cells. When using pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc. Tag fusion protein expression.

Expression vectors can be introduced into host cells by known methods such as electroporation, calcium phosphate method, liposome method, and DEAE-dextran method.

In order to isolate and purify a polypeptide of interest from host cells, known isolation procedures may be combined. Examples include denaturants such as urea, surfactant treatment, ultrasonic treatment, enzyme digestion, salting out, solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric point electrophoresis, ion Exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, etc., but not limited thereto.

Polypeptides obtained by the above methods also include polypeptides in the form of fusion proteins fused with other arbitrary proteins as described above. For example, a fusion protein fused with glutathione-S-transferase (GST), His tag, etc. can be exemplified. Therefore, such polypeptides in the form of fusion proteins are also included in the scope of the present invention. Furthermore, polypeptides expressed by transformed cells may undergo various modifications in the cells after translation. Such post-translationally modified polypeptides are also included in the scope of the present invention as long as they have immune-inducing activity. Examples of such translational modifications include removal of N-terminal methionine, N-terminal acetylation, addition of sugar chains, restriction degradation by intracellular proteases, myristylation, prenylation, phosphorylation, etc. .

＜Immune inducer＞

Administration of the polypeptide having immune-inducing activity of the present invention or an expression vector comprising a gene encoding the polypeptide to a cancer-bearing organism can cause regression of an already-generated tumor. In addition, tumorigenesis can be prevented by administering the above-mentioned polypeptide having immune-inducing activity or a gene encoding the polypeptide to a precancerous organism. Therefore, the polypeptide of the present invention or the gene encoding the polypeptide can be an active ingredient of an immunity-inducing agent.

Here, the terms "tumor" and "cancer" refer to malignant neoplasms and are used interchangeably. In this case, the target cancer is preferably a cancer expressing the SCD1 protein, and particularly preferably malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colon cancer, gastric cancer, malignant brain tumor, esophageal cancer, and lung cancer. .

The target animals are as described above, preferably mammals, more preferably mammals including primates, pets, domestic animals, sports animals, etc., further preferably humans, dogs or cats, particularly preferably humans.

The target cancer-affected individual (cancer patient when the individual is a human) is preferably a cancer-affected individual who expresses the SCD1 protein in vivo, and specifically, is preferably a cancer-affected individual screened by the cancer detection method described in WO2011/027807. Particularly preferably, it is a cancer that is screened because the expression level of the antibody against the SCD1 protein contained in a sample obtained from a subject organism is greater than that contained in a sample obtained from a healthy individual. individual. As the sample to be used for the screening of the target cancer individual, blood, serum, plasma, ascites, pleural effusion and other body fluids, tissues, and cells can be mentioned, but when screening by measuring the expression level of the antibody against the SCD1 protein, it is preferable Serum, plasma, ascites or pleural fluid.

The route of administration of the immunity-inducing agent of the present invention may be oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, and intraarterial administration is preferable. and. When the immune inducer is used for the purpose of treating cancer, it can also be administered to lymph nodes in the vicinity of the tumor to be treated in order to enhance the anticancer effect. The amount to be administered may be an amount effective for immunity induction, for example, when used for the treatment or prevention of cancer, any amount may be effective for the treatment and/or prevention of cancer. The amount effective for the treatment or prevention of cancer should be appropriately selected according to the size of the tumor, symptoms, body weight, volume, etc. of the target animal. When the target animal is a human, the effective amount per day is usually 0.0001-1000 μg, preferably 0.001-1000 μg, which can be 1 administered several times or divided into several times. It is preferably administered in fractions per day, every few days to several months. As will be specifically exemplified in Examples described later, the immunity-inducing agent of the present invention can shrink an already formed tumor. Therefore, since it can exert an anticancer effect on a small number of cancer cells in the early stage of development, it can prevent the onset and recurrence of cancer if it is used before the onset of cancer or after cancer treatment. That is, the immunity-inducing agent of the present invention is useful for both cancer treatment and prevention, and can be used as an active ingredient of a cancer treatment or prevention drug.

The immunity-inducing agent of the present invention contains the above-mentioned polypeptide of the present invention as an active ingredient, but may be composed of a single polypeptide or a combination of multiple polypeptides. By combining multiple types of polypeptides of the present invention, the immune-inducing activity (induction and activation of cytotoxic T cells) possessed by each polypeptide is enhanced, and cancer treatment or prevention can be achieved more effectively.

The immunity-inducing agent of the present invention can be used in combination with known peptides capable of inducing cytotoxic T cells. By combining the polypeptides of the present invention, the immune-inducing activity (induction and activation of cytotoxic T cells) possessed by each polypeptide is enhanced, and cancer treatment or prevention can be achieved more effectively. The "combination" in this case includes separate or simultaneous administration of the immunity-inducing agent of the present invention and a known peptide capable of inducing cytotoxic T cells. The term "separate administration" as used herein means that the immunity-inducing agent of the present invention and a known peptide capable of inducing cytotoxic T cells are administered separately with a time difference. The order of administration is not considered. On the other hand, "simultaneous administration" refers to pre-mixing the immunity-inducing agent of the present invention with a known peptide capable of inducing cytotoxic T cells and administering it in an integrated manner, or combining the immunity-inducing agent of the present invention with a known peptide. The peptides capable of inducing cytotoxic T cells were administered separately without time difference.

The immunity-inducing agent of the present invention can be used in combination with other immune-enhancing agents capable of enhancing the immune response in vivo. Other immunopotentiators may be included in the immune-inducing agent of the present invention, or may be administered to patients as another composition in combination with the immune-inducing agent of the present invention.

Examples of the aforementioned "other immunopotentiators" include adjuvants. Adjuvants can enhance the immune response by providing a reservoir of antigens (extracellular or intra-macrophage), activating macrophages, and stimulating specific lymphocytes, thus enhancing the anti-cancer effect. Therefore, when the immunity-inducing agent of the present invention is used as an active ingredient of a drug for treating or preventing cancer, the immunity-inducing agent preferably contains an adjuvant in addition to the above-mentioned polypeptide of the present invention as an active ingredient. A variety of adjuvants are known in the art and any adjuvant can be used. Specific examples of the adjuvant include MPL (SmithKline Beecham); the congener obtained after the lipopolysaccharide of Salmonella Minnesota (Salmonella minnesota) Re 595 is purified and acid hydrolyzed; QS21 (SmithKline Beecham); (Quillja saponaria) purified pure QA-21 saponins; DQS21 as described in PCT application WO 96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So, H.S., et al. al., 1997, Molecules and cells, 7:178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; Vitamin E; Montanide; Alum; al., 1995, Nature 374:546-549); polyinosin and its derivatives (polyICLC, etc.) and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol. Among them, Freund's incomplete adjuvant, Montanide, polyinosin and its derivatives, and CpG oligonucleotides are preferred. The mixing ratio of the adjuvant to the polypeptide is typically about 1:10 to 10:1, preferably about 1:5 to 5:1, more preferably about 1:1. However, the adjuvants are not limited to the above-mentioned examples, and adjuvants other than the above-mentioned ones known in the art may also be used when administering the immunity-inducing agent of the present invention (for example, refer to Goding, Monoclonal Antibodies: Principles and Practice, 2nd edition, 1986 ). Methods of preparing mixtures or emulsions of immunity-inducing agents and adjuvants are well known to those skilled in the art of vaccination.

In addition, as the above-mentioned other immunopotentiators, in addition to the above-mentioned adjuvants, factors that stimulate the immune response of the subject can also be used. For example, various cytokines having the property of stimulating lymphocytes and antigen-presenting cells can be used in combination with the immunity-inducing agent of the present invention as an immunopotentiator. Various cytokines capable of enhancing the immune response are known to those skilled in the art, and examples thereof include interleukin-12 (IL-12), GM-CSF, IL-18, Interferon alpha (IFN-alpha), interferon beta (IFN-beta), interferon omega (IFN-omega), interferon gamma (IFN-gamma), and Flt3 ligand, but not limited to these. Such factors can also be used as the above-mentioned immunopotentiator, and can be included in the immunity-inducing agent of the present invention, or administered to patients as another composition in combination with the immunity-inducing agent of the present invention.

＜Therapeutic or preventive drugs for cancer＞

The immunity-inducing agent of the present invention can be used as an active ingredient of a drug for treating or preventing cancer.

The drug for treating or preventing cancer can be formulated by appropriately mixing the immune inducer of the present invention with pharmacologically acceptable carriers, diluents, excipients and other additives suitable for each administration mode.

Formulation methods and additives that can be used are well known in the field of pharmaceutical preparations, and any methods and additives can be used. Specific examples of additives include diluents such as physiological buffers; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine, etc.; syrup, gelatin, acacia, sorbitol, polychloride, etc. Binders such as ethylene and tragacanth; lubricants such as magnesium stearate, polyethylene glycol, talc, and silicon dioxide, etc., but are not limited to these. Examples of the preparation form include oral preparations such as tablets, capsules, granules, powders, and syrups; parenteral preparations such as inhalants, injections, suppositories, and liquid preparations; and the like. These formulations can be produced by conventionally known production methods.

＜Antigen Presenting Cell＞

In addition, the polypeptide can be presented to the antigen-presenting cells by contacting the above-mentioned polypeptide with antigen-presenting cells in vitro. That is, the above-mentioned polypeptide (a) or (b) can be used as a treatment agent for antigen-presenting cells. Here, as antigen-presenting cells, dendritic cells or B cells retaining MHC class I and class II molecules can be preferably used. A variety of MHC class I and class II molecules have been identified and are well known. The MHC molecules in humans are called HLAs. HLA class I molecules include HLA-A, HLA-B, and HLA-C, and more specifically, HLA-A1, HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, HLA-Cw0602, etc. HLA class II molecules include HLA-DR, HLA-DQ, and HLA-DP, and more specifically, HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*0405, HLA-DRB1*0405, and HLA-DRB1*04. DRB1*07, HLA-DRB1*08, HLA-DRB1*11, HLA-DRB1*13, HLA-DRB1*15, HLA-DRB1*15, HLA-DQA1, HLA-DQB1, HLA-DPB1.

Dendritic cells or B cells retaining MHC class I or MHC class II molecules can be prepared from blood or the like by a known method. To this culture system, tumor-associated Peptides that induce tumor-specific dendritic cells.

By administering an effective amount of the dendritic cells, an immune response desired for cancer treatment can be induced. The cells used can be bone marrow provided by healthy people, umbilical cord blood, patient's own bone marrow, peripheral blood, etc., but the use of the patient's own cells is highly safe and can be expected to avoid serious side effects, so it is preferable. Peripheral blood or bone marrow may be any of fresh samples, cryopreserved samples, and cryopreserved samples. Peripheral blood may be cultured as whole blood or only leukocyte components may be separated and cultured, but the latter is preferred from the viewpoint of efficiency. Furthermore, monocytes can also be separated from the leukocyte component. In addition, in the case of bone marrow or umbilical cord blood as the source, all the cells constituting the bone marrow may be cultured, or mononuclear cells may be isolated therefrom and cultured. Monocytes, hematopoietic stem cells, immature dendritic cells, CD4-positive cells, and the like are contained in peripheral blood, its leukocyte components, and bone marrow cells. As long as the cytokines used are those whose safety and physiological activity have been confirmed, it does not matter whether they are natural type or gene recombinant type, and regardless of their production method, but they are preferably used in the minimum required amount to ensure medical use. standard of quality. The concentration of cytokines to be added is not particularly limited as long as it induces dendritic cells, but usually the total concentration of cytokines is preferably about 10 to 1000 ng/ml, more preferably about 20 to 500 ng/ml. Culturing can be performed using a known medium generally used for leukocyte culture. The culture temperature is not particularly limited as long as leukocytes can proliferate, but it is most preferably about 37° C., which is human body temperature. In addition, the gas environment during culture is not particularly limited as long as leukocytes can proliferate, but 5% CO ₂ aeration is preferred. In addition, the culture period is not particularly limited as long as the required number of cells can be induced, and it is usually carried out between 3 days and 2 weeks. Appropriate devices can be used for the isolation and culture of cells, but are preferably those that have been confirmed to be safe for medical use, and are stable and easy to operate. In particular, cell culture devices are not limited to common containers such as petri dishes, flasks, and culture bottles, and layered containers, multi-stage containers, roller bottles, spinner bottles, and bag culture devices can also be used , Hollow wire column, etc.

The method itself of bringing the above-mentioned polypeptide into contact with antigen-presenting cells in vitro (in vitro) can be performed by a known method. For example, it can be achieved by culturing antigen-presenting cells in a culture solution containing the above-mentioned polypeptide. The concentration of the peptide in the medium is not particularly limited, but is usually about 1 to 100 μg/ml, preferably about 5 to 20 μg/ml. The cell density during culture is not particularly limited, but is usually about 10 ³ to 10 ⁷ cells/ml, preferably about 5×10 ⁴ to 5×10 ⁶ cells/ml. Preferably, the culture is carried out in an environment of 37° C. and 5% CO ₂ according to conventional methods. In addition, the length of a peptide that can be presented on the surface of an antigen-presenting cell is usually about 30 amino acid residues at most. Therefore, although not particularly limited, in the case of contacting an antigen-presenting cell with the polypeptide in vitro, the polypeptide can be made to have a length of about 30 amino acid residues or less.

By culturing antigen-presenting cells in the presence of the above-mentioned polypeptide, the peptide is inserted into the MHC molecule of the antigen-presenting cell and presented on the surface of the antigen-presenting cell. Therefore, the use of the above-mentioned polypeptide enables the production of isolated antigen-presenting cells comprising a complex of the polypeptide and MHC molecules. Such antigen-presenting cells can present the polypeptide to T cells in vivo or in vitro (in vitro), induce and proliferate cytotoxic T cells or helper T cells specific to the polypeptide.

Cytotoxic T cells or helper T cells specific to the polypeptide can be induced and proliferated by contacting the antigen-presenting cells containing the complex of the polypeptide and MHC molecule prepared as described above with T cells in vitro. This can be performed by co-cultivating the above-mentioned antigen-presenting cells with T cells in a liquid medium. For example, it can be carried out by suspending antigen-presenting cells in a liquid medium, placing the obtained suspension in a container such as a well of a microplate, adding T cells thereto, and culturing. The mixing ratio of antigen-presenting cells and T cells during co-cultivation is not particularly limited, but usually the ratio of the number of cells is about 1:1 to 1:100, preferably about 1:5 to 1:20. The density of the antigen-presenting cells suspended in the liquid medium is not particularly limited, but is usually about 1 to 10 million cells/mL, preferably about 10,000 to 1 million cells/mL. Co-cultivation is preferably carried out in an environment of 37° C. and 5% CO ₂ according to conventional methods. The culture time is not particularly limited, but it is usually about 2 days to 3 weeks, preferably about 4 days to 2 weeks. Co-cultivation is preferably carried out in the presence of one or more interleukins such as IL-2, IL-6, IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5-20 U/mL, the concentration of IL-6 is usually about 500-2000 U/mL, and the concentration of IL-12 is usually 5-20 ng/mL around, but not limited to these. The above co-cultivation can also be repeated once or several times by adding fresh antigen-presenting cells. For example, the operation of discarding the culture supernatant after co-cultivation and adding a fresh suspension of antigen-presenting cells to further co-cultivate can be repeated once or several times. The conditions of each co-cultivation can be the same as above.

Cytotoxic T cells and helper T cells specific to the polypeptide are induced and proliferated by the above-mentioned co-cultivation. Thus, using the above-mentioned polypeptide, isolated T cells that selectively bind to the complex of the polypeptide and MHC molecule can be prepared.

As described in the Examples described later, the gene encoding the SCD1 protein (SCD1 gene) was expressed in malignant lymphoma tissue, malignant lymphoma cell, breast cancer tissue, breast cancer cell, liver cancer tissue, liver cancer cell, prostate cancer tissue, prostate cancer tissue, etc. cell, ovarian cancer tissue, ovarian cancer cell, kidney cancer tissue, kidney cancer cell, colorectal cancer tissue, colorectal cancer cell, gastric cancer tissue, gastric cancer cell, malignant brain tumor tissue, malignant brain tumor cell, esophageal cancer tissue, esophageal cancer cell, Specifically expressed in lung cancer tissues and lung cancer cells. Therefore, it is considered that the SCD1 protein exists significantly more in these cancer types than in normal cells. A part of the SCD1 protein present in cancer cells is presented by MHC molecules on the surface of the cancer cells, and when the cytotoxic T cells or helper T cells prepared as described above are administered in vivo, the cytotoxic T cells express the SCD1 protein Part of the target is to poison cancer cells or enhance the cytotoxic activity of cytotoxic T cells. In addition, since the antigen-presenting cells presenting the above-mentioned polypeptide can also induce and proliferate cytotoxic T cells and helper T cells specific to the polypeptide in vivo, it is also possible to generate Toxic T cells injure cancer cells, or enhance the cytotoxic activity of cytotoxic T cells. That is, the above-mentioned cytotoxic T cells, helper T cells, and above-mentioned antigen-presenting cells prepared using the above-mentioned polypeptides are also useful as a therapeutic or preventive drug for cancer like the immunity-inducing agent of the present invention.

When the above-mentioned isolated antigen-presenting cells and isolated T cells are administered to a living body, in order to avoid an immune response in the living body that attacks these cells as foreign substances in the living body, it is preferable that these isolated cells be obtained from a patient receiving treatment. The collected antigen-presenting cells or T cells are prepared as described above using the polypeptide of (a) or (b).

The route of administration of the drug for treating or preventing cancer containing antigen-presenting cells or isolated T-cell antigens as an active ingredient is preferably parenteral administration such as intravenous administration or intraarterial administration. In addition, the dose to be administered is appropriately selected depending on symptoms, purpose of administration, etc., and is usually 1 to 10 trillion cells, preferably 1 million to 1 billion cells, and is preferably administered every few days or months. The preparation may be a suspension prepared by suspending cells in, for example, physiological buffered saline, or may be used in combination with other anticancer agents, cytokines, and the like. In addition, one or two or more additives known in the pharmaceutical field may be added.

＜Gene Vaccine＞

In addition, according to the present invention, by expressing the polynucleotide encoding the polypeptide (a) or (b) above in the body of the subject animal, it is also possible to induce immunity, that is, to induce antibody production and cytotoxic T cells in the living body, The same effect as that of administering the polypeptide is obtained. That is, the immunity-inducing agent of the present invention may contain, as an active ingredient, a recombinant vector comprising a polynucleotide encoding the polypeptide of (a) or (b) above and capable of expressing the polypeptide in vivo. As shown in the following examples, such recombinant vectors capable of expressing antigenic polypeptides are also called "genetic vaccines".

The carrier used to make the gene vaccine is not particularly limited as long as it can be expressed in the target animal cells (preferably mammalian cells). Any carrier. In addition, polynucleotides such as DNA and RNA encoding the above-mentioned polypeptides can be easily prepared by conventional methods as described above. Alternatively, the polynucleotide can be inserted into a vector by using methods well known to those skilled in the art.

The route of administration of the genetic vaccine is preferably parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, and intraarterial administration. About 0.1 μg to 100 mg of body weight, preferably about 1 μg to 10 mg

Examples of methods using viral vectors include inserting a polynucleotide encoding the above-mentioned polypeptide into RNA such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, etc. A method of infecting a target animal with a virus or a DNA virus. Among them, methods using retrovirus, adenovirus, adeno-associated virus, vaccinia virus, etc. are particularly preferable.

Other methods include a method in which an expression plasmid is directly administered intramuscularly (DNA vaccine method), liposome method, lipofection (Lipofectin) method, microinjection method, calcium phosphate method, electroporation method, etc. , DNA vaccine method and liposome method are particularly preferred.

In order to make the gene encoding the above-mentioned polypeptide used in the present invention actually function as a drug, there are in vivo methods of directly introducing the gene into the body, collecting certain cells from the target animal, introducing the gene into the cells in vitro, and An ex vivo method in which the cells are returned to the body, more preferably an in vivo method.

When administered by an in vivo method, it can be administered by an appropriate administration route according to the disease, symptom, etc. to be treated. For example, intravenous, arterial, subcutaneous, intramuscular and the like can be administered. When administered by in vivo methods, for example, liquid preparations and other preparations can be used, and injections containing the DNA encoding the above-mentioned peptide of the present invention as an active ingredient are usually prepared, and conventional carriers can be added as needed. In addition, liposomes or membrane fusion liposomes (Sendai virus (HVJ)-liposomes, etc.) containing the DNA can be in the form of liposome preparations such as suspensions, cryogens, centrifuged concentrated cryogens, and the like.

In addition, in the present invention, when referring to the "base sequence shown in SEQ ID NO: 1", in addition to the base sequence actually shown in SEQ ID NO: 1, a complementary sequence is also included. Therefore, when referring to a "polynucleotide having a base sequence shown in SEQ ID NO: 1", it includes a single-stranded polynucleotide having a base sequence actually shown in SEQ ID NO: 1, a polynucleotide having a base sequence complementary thereto Single-stranded polynucleotides, and double-stranded polynucleotides consisting of them. When preparing a polynucleotide encoding a polypeptide used in the present invention, any base sequence can be appropriately selected, and such selection can be easily performed by those skilled in the art.

Example

Hereinafter, based on an Example, this invention is demonstrated more concretely.

<Example 1: Expression analysis in each tissue>

(1) Analysis of SCD1 gene expression in each cancer cell line

The gene sequence (SEQ ID NO: 1) encoding the amino acid sequence of human SCD1 protein was obtained from Gene Bank. The expression of the obtained genes in various human cell lines was studied by RT-PCR (Reverse Transcription-PCR) method. The reverse transcription reaction was performed as follows. That is, total RNA was extracted from 50 to 100 mg of each tissue and 5 to 10×10 ⁶ cells of each cell line using TRIZOL reagent (manufactured by Life Technologies) according to the attached protocol. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Life Technologies) according to the attached protocol. For cDNA of human normal tissues (brain, hippocampus, testis, colon, placenta), GenePool cDNA (manufactured by Life Technologies), QUICK-Clone cDNA (manufactured by Crontec), and Large-Insert cDNA Library (manufactured by Crontec) were used. Using primers specific to the obtained gene (the base sequences of the primers are described in SEQ ID NOs: 49 and 50), a PCR reaction was carried out as follows. That is, each reagent and the attached buffer were added so that the total amount was 25 μl by adding 0.25 μl of the sample prepared by the reverse transcription reaction, 2 μM each of the above primers, 0.2 mM each dNTP, and 0.65 U ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.). , using a Thermal Cycler (BIO RAD), the cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute was repeated 30 times. For comparison purposes, primers specific to the housekeeping gene GAPDH gene were also used simultaneously (the base sequences of the human GAPDH primers are described in SEQ ID NOs: 51 and 52).

The results are shown in Figure 1, the human SCD1 gene in most cancer cell lines, that is, malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, gastric cancer, malignant brain tumors, esophageal cancer and lung cancer. expression detected.

(2) Expression of SCD1 protein in human cancer tissue (immunohistochemical staining)

Immunohistochemical staining was carried out using 72 samples of cancer tissues embedded in paraffin-embedded multi-cancer tissue array (manufactured by BIOMAX). After the human cancer tissue array was treated at 60° C. for 3 hours, it was placed into a staining bottle filled with xylene, and the operation of replacing xylene every 5 minutes was repeated 3 times. Next, the same operation was performed using ethanol and PBS-T instead of xylene. Human cancer tissue arrays were added to staining bottles filled with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween20, treated at 125° C. for 5 minutes, and then left to stand at room temperature for more than 40 minutes. Wipe off excess water around the section with a Kimwipe (dust-free wiping paper), surround it with DAKOPEN, and add an appropriate amount of Peroxidase Block (manufactured by DAKO) dropwise. After standing at room temperature for 5 minutes, they were put into staining bottles filled with PBS-T, and the operation of replacing PBS-T every 5 minutes was performed 3 times. As a blocking solution, a PBS-T solution containing 10% FBS was added and allowed to stand at room temperature in a humid box for 1 hour. Next, a commercially available rabbit polyclonal antibody (Sigma) that reacts with the SCD1 protein was added at 10 μg/mL in PBS-T solution containing 5% FBS, and left to stand at 4° C. overnight in a humid chamber. After washing with PBS-T three times for 10 minutes, an appropriate amount of Peroxidase Labeled Polymer Conjugated (manufactured by DAKO) was added dropwise, and left to stand at room temperature in a humid chamber for 30 minutes. After washing with PBS-T for 3 times for 10 minutes, add DAB color developing solution (manufactured by DAKO Co., Ltd.), leave at room temperature for about 10 minutes, discard the color developing solution, and wash with PBS-T for 3 times for 10 minutes. Rinse with distilled water, put in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 1 minute each in turn, and then let stand overnight in xylene. The slide glass was taken out, sealed with Glycergel Mounting Medium (manufactured by DAKO), and observed.

As a result, strong expression of the SCD1 protein was confirmed in most verified cancers, malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, gastric cancer, malignant brain tumor, esophageal cancer, and lung cancer.

<Example 2: Induction of peptide epitope-reactive CD8-positive T cells>

(1) Prediction of peptide motifs binding to HLA-A0201 and HLA-A24

Information on the amino acid sequence of the human SCD1 protein shown in SEQ ID NO: 2 was obtained from GenBank. To predict the binding motifs of HLA-A0201 and HLA-A24, human SCD1 protein 21 kinds of polypeptides composed of the amino acid sequences shown in the sequence numbers 3 to 23 that are expected to bind to the HLA-A0201 molecule and 21 kinds of polypeptides shown in the sequence numbers 24 to 36 that are expected to bind to the HLA-A24 molecule were selected. There are 13 kinds of polypeptides composed of amino acid sequences. All the selected peptides were commissioned to be synthesized by the custom peptide synthesis service of Grainer Japan Co., Ltd. In addition, the quality of the synthesized peptides is guaranteed by HPLC analysis and mass spectrometry.

(2) Induction of peptide epitope-reactive CD8-positive T cells

Peripheral blood was isolated from HLA-A0201 positive healthy people, layered on Lymphocyte separation medium (OrganonpTeknika, Durham, NC), and centrifuged at 1500 rpm for 20 minutes at room temperature. The fraction containing PBMCs was recovered, and washed 3 times (or more than 3 times) in cold phosphate buffered saline to obtain PBMCs. The obtained PBMCs were suspended in 20 mL of AIM-V medium (manufactured by Life Technologies), and allowed to adhere to a culture flask (manufactured by Falcon) at 37°C and 5% CO ₂ for 2 hours. Non-attached cells are used to make T cells, and attached cells are used to make dendritic cells.

Adherent cells were cultured in AIM-V medium in the presence of IL-4 (1000 U/mL) and GM-CSF (1000 U/mL). After 6 days, it was replaced with supplemented with IL-4 (1000U/mL), GM-CSF (1000U/mL), IL-6 (1000U/mL, manufactured by Genzyme), IL-1β (10ng/mL, manufactured by Genzyme) and The AIM-V medium of TNF-α (10 ng/mL, manufactured by Genzyme) was further cultured for 2 days, and the resulting non-adherent cell population was used as dendritic cells.

The prepared dendritic cells were suspended in AIM-V medium at a cell density of 1×10 ⁶ cells/mL, and the peptide selected in (1) above that was expected to bind to HLA-A0201 molecules was added at a concentration of 10 μg/mL , using a 96-well plate and cultured for 4 hours at 37°C and 5% CO ₂ . After culturing, X-rays were irradiated (3000 rad), washed with AIM-V medium, and treated with 10% human AB serum (manufactured by Nabi), IL-6 (1000 U/mL) and IL-12 (10 ng/mL, Genzyme Co., Ltd.) AIM-V medium, and each 1×10 ⁵ cells were added to each well of a 24-well plate. Further, 1×10 ⁶ cells were added to each well of the prepared T cell population, and cultured at 37° C. and 5% CO ₂ . After 7 days, each culture supernatant was discarded, and dendritic cells treated with each peptide obtained in the same manner as above and irradiated with X-rays were treated with 10% human AB serum (manufactured by Nabi), IL-7 (10 U/mL , manufactured by Genzyme) and IL-2 (10 U/mL, manufactured by Genzyme) in AIM-V medium (cell density: 1×10 ⁵ cells/mL), each well was added to each well of a 24-well plate. 1 x 10 ⁵ cells were further cultured. The same operation was repeated at 7-day intervals, and after four repetitions, the stimulated T cells were recovered, and the induction of CD8-positive T cells was confirmed by flow cytometry.

In addition, as a negative control, a peptide (SEQ ID NO: 46) of a sequence outside the scope of the present invention and an SCD1 protein composed of the amino acid sequence shown in SEQ ID NO: 2 produced based on Example 3 of WO2012/157736 were used as a comparative example to carry out The same processing as above.

In addition, for peptides expected to bind to HLA-A24 molecules, peptide epitope-reactive CD8-positive peptides were also tested in the same manner as above using dendritic cells and T cell populations induced from the peripheral blood of HLA-A24-positive normal humans. Induction of T cells. In addition, a peptide (SEQ ID NO: 47) of a sequence outside the scope of the present invention was used as a negative control, and the above-mentioned SCD1 protein having the amino acid sequence shown in SEQ ID NO: 2 was used as a comparative example to perform the same treatment.

<Example 3: Identification of cytotoxic T cell antigen epitopes>

(1) IFN-γ production ability

In order to examine the specificity of the T cells induced in Example 2(2) against epitope peptides and proteins, respectively, various polypeptides were pulsed to dendritic cells expressing the HLA-A0201 molecule. The dendritic cells were prepared by adding each polypeptide to AIM-V medium at a concentration of 10 μg/mL and culturing at 37° C. and 5% CO ₂ for 4 hours. In addition, as various polypeptides, each polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 23 expected to bind to the HLA-A0201 molecule, the negative control polypeptide (SEQ ID NO: 46), and the SCD1 protein composed of the amino acid sequence represented by SEQ ID NO: 2 were used . Add 5×10 ³ T cells to 5×10 ⁴ pulsed dendritic cells, and culture them in a 96-well plate in AIM-V medium containing 10% human AB serum for 24 hours. The supernatant after culture was taken, and the production amount of IFN-γ was determined by ELISA method.

As a result, bands 4 to 2 of dendritic cells pulsed with the polypeptides represented by the amino acid sequences of SEQ ID NOs. 24 significantly high IFN-γ production was confirmed (Fig. 2). From the results, it was found that the peptides of SEQ ID NOs. 3 to 23 are T cell epitope peptides capable of specifically stimulating the proliferation of HLA-A0201-positive CD8-positive T cells and inducing the production of IFN-γ. Furthermore, it was found that the production of IFN-γ using these peptides was also significantly higher than that produced by T cells stimulated with the full-length SCD1 protein (band 3) consisting of the amino acid sequence shown in SEQ ID NO: 2. That is, the polypeptides of SEQ ID NOs. 3 to 23 showed remarkably high immunity-inducing activity. In addition, although the amino acid sequence of the full-length SCD1 protein shown in SEQ ID NO: 2 includes the above-mentioned SEQ ID NOs 3 to 23 having immune-inducing activity, the IFN-γ produced by T cells stimulated by the full-length SCD1 protein of SEQ ID NO: 2 production is low. This is considered to be because the amino acid sequence of the full-length SCD1 protein also contains a large number of sequences that suppress immune-inducing activity, and thus does not exhibit sufficient immune-inducing activity.

Furthermore, in the same manner as above, in order to investigate the specificity for the peptide epitope of the peptide epitope-reactive CD8-positive T cells induced by using the polypeptide represented by the amino acid sequence of SEQ ID NO: 24 to 36 in Example 3 (2), according to The above method is aimed at the expression of the full-length SCD1 protein shown in the amino acid sequence of sequence number 24-36 (band 4-16), the negative control polypeptide shown in the amino acid sequence of sequence number 47, and the amino acid sequence of sequence number 2 by ELISA method Dendritic cells with HLA-A24 molecules, and the production of IFN-γ by T cells were measured.

As a result, compared with band 1 of dendritic cells not pulsed with the polypeptide and band 2 using the negative control polypeptide, bands 4 to 16 of the dendritic cells pulsed with the polypeptides of SEQ ID NOs. 24 to 36, Significant IFN-γ production was confirmed in the culture supernatant (Fig. 3).

From these results, it was found that the polypeptides of SEQ ID NOs. 24 to 36 are T cell epitope peptides capable of specifically stimulating the proliferation of HLA-A24-positive CD8-positive T cells and inducing the production of IFN-γ. Furthermore, it was found that the production amount of IFN-γ using these polypeptides was significantly higher than that produced by T cells stimulated with the full-length SCD1 protein shown in the amino acid sequence of SEQ ID NO: 2. For the same reason as above, it is considered that the full-length SCD1 protein does not exhibit sufficient immunity-inducing activity.

(2) Cytotoxicity evaluation

Next, it was studied whether the polypeptides represented by the amino acid sequences of the sequence numbers 3-23 used in the present invention were presented on HLA-A0201 molecules on HLA-A0201-positive tumor cells expressing human SCD1 protein, and whether the polypeptides shown by the amino acid sequences of the present invention were presented on HLA-A0201 molecules. Whether the CD8-positive T cells stimulated by polypeptides poison HLA-A0201-positive tumor cells expressing human SCD1 protein, and whether they significantly poison tumor cells compared with CD8-positive T cells stimulated by SCD1 protein.

Human glioma (malignant brain tumor) cell line U251 cell line confirmed to express human SCD1 protein, leukemia cell line THP1 liver cancer cell line SK-Hep-1, breast cancer cell line MCF7, ovarian cancer cell line OVCAR3, kidney cancer cell line 10 ^{and 6} cell lines of strain A498, colorectal cancer cell line HCT116, gastric cancer cell line AGS, and lung cancer cell line NCI-H522 (purchased from JCRB, RIKEN, and ATCC) were respectively collected in 50mL centrifuge tubes, and added 100 μCi of Chromium 51, incubate at 37°C for 2 hours. Then, wash 3 times with RPMI medium (manufactured by Kibuco) containing 10% fetal bovine serum (hereinafter referred to as FBS, manufactured by Kibuco), add 1× ¹⁰ to each well of a 96-well V-bottom plate, and further 5×10 ⁴ polypeptides represented by the amino acid sequences of SEQ ID NO: 3-23, the negative control polypeptide (SEQ ID NO: 46) and the amino acid sequence of SEQ ID NO: 2 were added to it, respectively, suspended in RPMI medium containing 10% FBS HLA-A0201-positive CD8-positive T cells induced by stimulation with the indicated full-length SCD1 protein were cultured at 37° C. and 5% CO ₂ for 4 hours. After culturing, the amount of chromium 51 in the culture supernatant released from the toxic tumor cells was measured to calculate the cytotoxic activity of CD8-positive T cells induced by the stimulation of each polypeptide and protein.

As a result, it was found that HLA-A0201-positive CD8-positive T cells induced by stimulation with the polypeptides represented by the amino acid sequences of SEQ ID NOs: 3 to 23 had significant cytotoxic activity against all of the above-mentioned cells. As a representative example, Figures 4A and 4B show the results of cytotoxic activity against U251 cells and SK-Hep-1 cells, respectively. Compared with CD8 positive T cells (band 3) stimulated by the full-length SCD1 protein, CD8-positive T cells stimulated by the polypeptides represented by the amino acid sequences of SEQ ID NO: 3-23 are more effective against U251 cells and SK-Hep-1 cells showed significantly high cytotoxic activity. On the other hand, the negative control polypeptide (lane 2) induced CD8-positive T cells to the same extent as Mock (lane 1), and did not show cytotoxic activity. This result suggests that the polypeptides of sequence numbers 3 to 23 used in the present invention are presented to HLA-A0201 molecules on HLA-A0201-positive tumor cells expressing human SCD1 polypeptides, and furthermore, the polypeptides of the present invention have the ability to induce toxicity CD8-positive cytotoxic T cell capacity of tumor cells. In addition, although the amino acid sequence of the full-length SCD1 protein includes SEQ ID NOs 3 to 23, the cytotoxic activity of CD8-positive T cells stimulated by the polypeptides of SEQ ID NOs 3 to 23 was significantly weaker (bands 3, 4 to 24). This is considered to be because the amino acid sequence of the SCD1 protein contains a large number of sequences that suppress immune-inducing activity, and thus cannot induce T cells having strong cytotoxic activity.

Similarly, it is investigated whether the polypeptides of SEQ ID NOs. 24-36 are presented on HLA-A24 molecules on HLA-A24-positive tumor cells expressing human SCD1 protein, and whether CD8-positive T cells stimulated by the polypeptides of the present invention can poison Whether HLA-A24-positive tumor cells expressing human SCD1 protein are significantly toxic to tumor cells compared with CD8-positive T cells stimulated by SCD1 protein.

Human glioma cell line KNS-42, liver cancer cell line SK-Hep1, kidney cancer cell line Caki1, colorectal cancer cell line SW480, gastric cancer cell line MKN45, prostate cancer cell line that are HLA-A24 positive and express human SCD1 protein PC3, breast cancer cell line ZR75-1 (purchased from JCRB, Institute of Physical Chemistry and ATCC) were mixed with chromium 51, and the polypeptide shown in the amino acid sequence of sequence numbers 24 to 36, the negative control polypeptide (sequence number 47), and The amount of chromium 51 in the culture supernatant released from the poisoned cells when the HLA-A24 positive CD8 positive T cells induced by the stimulation of the full-length SCD1 protein.

As a result, it was found that HLA-A24-positive CD8-positive T cells stimulated by the polypeptides represented by the amino acid sequences of SEQ ID NOs: 24 to 36 had significant cytotoxic activity against all cancer cells used, which was usually unexpected. As a representative example, Figures 5A and 5B show the results of cytotoxic activity against SW480 cells and ZR75-1 cells, respectively. Compared with the CD8 positive T cells stimulated by the full-length SCD1 protein (band 3), the CD8-positive T cells stimulated by the polypeptides represented by the amino acid sequences of SEQ ID NO: 24-36 are more effective against SW480 cells. , and ZR75-1 cells showed significantly high cytotoxic activity. On the other hand, CD8-positive T cells induced by the polypeptide of the negative control were at the same level as Mock (lane 1), and showed no cytotoxic activity (lane 2). Therefore, SEQ ID NOs. 24 to 36 were presented on HLA-A24 molecules on HLA-A24-positive cells expressing human SCD1 protein, and the results showed that the polypeptide of the present invention has the ability to induce CD8-positive cytotoxic T cells capable of poisoning such cells Ability.

On the other hand, when the above-mentioned cancer cells were exposed to the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 36 and the full-length SCD1 protein consisting of the amino acid sequence represented by SEQ ID NO: 2, the cancer cells did not die at all. It was also confirmed that these polypeptides did not directly kill cancer cells.

The cytotoxic activity was shown by mixing 5×104 CD8-positive ^T cells induced by stimulation with each polypeptide used in the present invention and 1× ¹⁰³ tumor cells each incorporating Chromium 51 and culturing them for 4 hours, as described above. , the amount of chromium 51 released into the medium after culture was measured, and the result of the cytotoxic activity of CD8-positive T cells to each tumor cell (called target cell) calculated by the following calculation formula *.

*Formula: cytotoxic activity (%) = free amount of chromium 51 released from target cells when CD8 positive T cells were added ÷ free amount of chromium 51 released from target cells added with 1N hydrochloric acid × 100

<Example 4: Induction of peptide epitope-reactive CD4-positive T cells derived from SCD1 protein>

In order to predict the epitope of CD4-positive T cells, the amino acid sequence of human SCD1 protein was analyzed using the computer prediction program of SYFPE ITH I algorithm (written by Rammensee), and nine peptides indicated by sequence numbers 37 to 45 predicted to be HLA class II binding peptides were selected. . All the selected peptides were commissioned to be synthesized by the custom peptide synthesis service of Grainer Japan Co., Ltd.

Peripheral blood was isolated from HLA-DRB1*04-positive healthy people, layered on Lymphocyte separation medium (manufactured by OrganonpTeknika), and centrifuged at 1500 rpm for 20 minutes at room temperature. The fraction containing PBMCs was recovered, and washed 3 times (or more than 3 times) in cold phosphate buffered saline to obtain PBMCs. The obtained PBMCs were suspended in 20 mL of AIM-V medium (manufactured by Life Technologies), and allowed to adhere to a culture flask (manufactured by Falcon) at 37°C and 5% CO ₂ for 2 hours. Non-attached cells are used to make T cells, and attached cells are used to make dendritic cells.

On the other hand, attached cells were cultured in the presence of IL-4 (1000 U/mL) and GM-CSF (1000 U/mL) in AIM-V medium. After 6 days, it was replaced with supplemented with IL-4 (1000U/mL), GM-CSF (1000U/mL), IL-6 (1000U/mL, manufactured by Genzyme), IL-1β (10ng/mL, manufactured by Genzyme) and The AIM-V medium of TNF-α (10 ng/mL, manufactured by Genzyme) was further cultured for 2 days, and the resulting non-adherent cell population was used as dendritic cells.

The prepared dendritic cells were suspended in AIM-V medium at a cell density of 1×10 ⁶ cells/mL, and the polypeptides of sequence numbers 37-45 and the negative control polypeptide (sequence number 48) and the SCD1 protein consisting of the amino acid sequence shown in SEQ ID NO: 2 were cultured in a 96-well plate at 37° C. and 5% CO ₂ for 4 hours. After culturing, X-rays were irradiated (3000 rad), washed with AIM-V medium, and treated with 10% human AB serum (manufactured by Nabi), IL-6 (1000 U/mL) and IL-12 (10 ng/mL, Genzyme Co., Ltd.) AIM-V medium was suspended, and 1×10 ⁵ cells were added to each well of a 24-well plate. Further, 1×10 ⁶ cells were added to each well of the prepared T cell population, and cultured at 37° C. and 5% CO ₂ . After 7 days, each culture supernatant was discarded, and the dendritic cells irradiated with X-rays after treatment with each peptide and SCD1 protein obtained in the same manner as above were treated with 10% human AB serum (manufactured by Nabi) and IL-2 ( 10 U/mL, manufactured by Genzyme) in AIM-V medium was suspended, and 1×10 ⁵ cells were added to each well of a 24-well plate for further culture. The same operation was repeated 4 times every 7 days, and the stimulated T cells were recovered, and the induction of CD4-positive T cells was confirmed by flow cytometry. As a result, the induced proliferation of T cells in each well was confirmed.

<Example 5: Identification of helper T cell antigen epitope derived from SCD1 protein that stimulates HLA-DRB1*04-positive CD4-positive T cells>

In order to examine the specificity of the CD4-positive T cells induced in Example 4 above to each peptide protein, PBMCs expressing HLA-DRB1*04 molecule were pulsed with each peptide. The PBMCs were prepared by adding each polypeptide to AIM-V medium at a concentration of 10 μg/mL and culturing at 37° C. and 5% CO ₂ for 4 hours. In addition, as various polypeptides, each polypeptide represented by the amino acid sequence of SEQ ID NO: 37 to 45, the negative control polypeptide (SEQ ID NO: 48), and the full-length SCD1 protein consisting of the amino acid sequence represented by SEQ ID NO: 2 were used. Add 5×10 ⁴ CD4 positive T cells to 5×10 ⁴ pulsed PBMCs, and culture them in a 96-well plate for 24 hours in AIM-V medium containing 10% human AB serum. The supernatant after culture was taken, and the production amount of IFN-γ was determined by ELISA method.

As a result, 1000 pg/mL or more of IFN-γ was produced in the culture supernatant of the wells using PBMC pulsed with the respective peptides of SEQ ID NOs. 37 to 45. On the other hand, almost no IFN-γ production was observed in the culture supernatant of the wells of dendritic cells (Mock) using only the negative control polypeptide and the non-pulsed polypeptide. Therefore, it was found that various polypeptides represented by the amino acid sequences of SEQ ID NOs: 37 to 45 are T cell epitope peptides capable of specifically stimulating proliferation of HLA-DRB1*04 positive CD4 positive T cells and inducing IFN-γ production. In addition, although the amino acid sequence of the full-length SCD1 protein contained the aforementioned SEQ ID NOs. 37-45 having immune-inducing activity, the production amount of IFN-γ in the culture supernatant of the wells of PBMC cells pulsed with the full-length SCD1 protein was extremely low. few. This is considered to be because the amino acid sequence of the SCD1 protein contains a large number of sequences that suppress immune-inducing activity, and thus does not exhibit sufficient immune-inducing activity.

Next, it was investigated whether the polypeptide of SEQ ID NO: 37-45, which has the ability to stimulate the proliferation of HLA-DRB1*04 positive T cells, is an epitope presented on HLA-DR by natural processing of SCD1 protein in antigen-presenting cells . The lysate of HEK293 cells (purchased from ATCC) transiently expressing the SCD1 protein was added to immature dendritic cells and digested to mature the dendritic cells, and the polypeptides of SEQ ID NOs: 37-45, Whether the T cells stimulated by the negative control polypeptide and SCD1 protein are stimulated by the dendritic cells. Peripheral blood was isolated from HLA-DRB1*04 positive healthy people, stacked on Lymphocyte separation medium, and centrifuged at 1500 rpm for 20 minutes at room temperature. The fractions containing PBMCs were harvested and washed 3 times (or more than 3 times) in cold phosphate buffered saline to obtain PBMCs. The obtained PBMCs were suspended in 20 mL of AIM-V medium (manufactured by Life Technologies Co., Ltd.), and allowed to adhere in a culture flask (Falcon) for 2 hours at 37°C and 5% CO ₂ . The immature dendritic cells were prepared by culturing in V medium for 6 days in the presence of IL-4 (1000 U/mL) and GM-CSF (1000 U/mL). Add the above lysate to 5× ¹⁰⁵ immature dendritic cells, add IL-4 (1000U/mL), GM-CSF (1000U/mL), IL-6 (1000U/mL), IL -1β (10ng/mL) and TNF-α (10ng/mL) were cultured in AIM-V medium for 2 days. The cultured dendritic cells were irradiated with X-rays (3000rad), washed with AIM-V medium, suspended in AIM-V medium containing 10% human AB serum, and placed in each well of a 96-well plate. Add 3.3 x ¹⁰⁴ each. Add 5×10 ⁴ T cells stimulated by the negative control polypeptide of sequence numbers 37-45 and SCD1 protein, and culture at 37° C. and 5% CO ₂ for 24 hours. The supernatant after culture was taken, and the production amount of IFN-γ was determined by ELISA method.

As a result, as shown in FIG. 6 , it was found that the T cells with bands 4 to 12 stimulated by the polypeptides of SEQ ID NOs. 37 to 45 produced IFN-γ by stimulation of SCD1 protein-added dendritic cells. On the other hand, almost no IFN-γ production was found in band 2 stimulated by the negative control polypeptide and band 1 not stimulated by the polypeptide. Therefore, it was clarified that the polypeptides of SEQ ID NOs. 37 to 45 are epitopes of the SCD1 protein that are naturally processed in antigen-presenting cells and presented on HLA-DR. In addition, band 3 of the full-length SCD1 protein was also pulsed in this experiment, and the production of IFN-γ was extremely small. It is considered that the amino acid sequence of the full-length SCD1 protein does not exhibit sufficient immune-inducing activity because a large number of sequences that suppress immune-inducing activity are included.

industry availability

The immunity-inducing agent of the present invention comprising a polypeptide exhibiting antitumor activity against various cancers is useful for the treatment or prevention of cancer, or for the detection of cancer.

All publications, patents, and patent applications cited in this specification are directly incorporated by reference into this specification.

Claims (12)

1. The immune inducer, as an active ingredient, comprising: At least one polypeptide consisting of any one of the amino acid sequences shown in SEQ ID NO: 37-45, capable of binding to MHC class II molecules and having immune-inducing activity, or A recombinant vector comprising at least one polynucleotide encoding any of the polypeptides and capable of expressing the polypeptide in vivo. 2. The immunity-inducing agent according to claim 1, which is used as an active ingredient of a drug for treating or preventing cancer. 3. The immunity-inducing agent according to claim 2, wherein the cancer expresses the SCD1 protein. 4. The immunity-inducing agent according to claim 3, wherein the cancer is malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colon cancer, stomach cancer, malignant brain tumor, esophagus cancer or lung cancer. 5. The immunity-inducing agent according to any one of claims 1 to 4, further comprising an immunity-enhancing agent. 6. An isolated antigen-presenting cell comprising a complex of the polypeptide having immune-inducing activity and MHC molecules according to claim 1. 7. An isolated T cell capable of selectively binding the complex of the polypeptide having immune-inducing activity and MHC molecule as claimed in claim 1. 8. A polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NO: 37-45, capable of binding to MHC class II molecules and having immune-inducing activity. 9. A drug for treating or preventing cancer, comprising as an active ingredient at least one selected from the group consisting of the following (i) to (iv), (i) at least one polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NO: 37-45, capable of binding to MHC class II molecules and having immune-inducing activity; (ii) a recombinant vector comprising at least one polynucleotide encoding any of the polypeptides and capable of expressing the polypeptide in vivo; (iii) an isolated antigen-presenting cell comprising a complex of any of said polypeptides with an MHC molecule; and (iv) isolated T cells specific for any of said polypeptides. 10. The drug for treating or preventing cancer according to claim 9, wherein the cancer expresses the SCD1 protein. 11. The use of one or more selected from the group consisting of the following (i) to (iv) for the manufacture of a drug for the treatment or prevention of cancer, (i) at least one polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NO: 37-45, capable of binding to MHC class II molecules and having immune-inducing activity; (ii) a recombinant vector comprising at least one polynucleotide encoding any of the polypeptides and capable of expressing the polypeptide in vivo; (iii) an isolated antigen-presenting cell comprising a complex of any of said polypeptides with an MHC molecule; and (iv) isolated T cells specific for any of said polypeptides. 12. The use according to claim 11, wherein the cancer is a cancer expressing the SCD1 protein.

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